Chemokine receptor homo- or heterodimerization activates distinct signaling pathways

Chemokine receptors of both the CC and CXC families have been demonstrated to undergo a ligand-mediated homodimerization process required for Ca2+ flux and chemotaxis. We show that, in the chemokine response, heterodimerization is also permitted between given receptor pairs, specifically between CCR2 and CCR5. This has functional consequences, as the CCR2 and CCR5 ligands monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cell-expressed and secreted) cooperate to trigger calcium responses at concentrations 10- to 100-fold lower than the threshold for either chemokine alone. Heterodimerization results in recruitment of each receptor-associated signaling complex, but also recruits dissimilar signaling path ways such as G(q/11) association, and delays activation of phosphatidyl inositol 3-kinase. The consequences are a pertussis toxin-resistant Ca2+ flux and trig gering of cell adhesion rather than chemotaxis. These results show the effect of heterodimer formation on increasing the sensitivity and dynamic range of the chemokine response, and may aid in understanding the dynamics of leukocytes at limiting chemokine concentrations in vivo.     

Kinetic analysis of estrogen receptor homo- and heterodimerization in vitro

The coexistence of ERalpha and ERbeta suggests that active receptor complexes are present as homo- or heterodimers. In addition each of three forms of active receptors may trigger different cellular responses. A real-time biosensor based on surface plasmon resonance was used as instrument to determine binding kinetics of homo- and heterodimerization of estrogen receptor alpha and beta. Partially purified full-length estrogen receptor alpha was expressed intracellularly as a C-terminal fusion to a hexa-histidine tag using the baculovirus-expression system. Purified estrogen receptor alpha and beta without tags were used as partners in the dimerization process. An association rate constant of 3.6 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the homodimer formation of ERalpha and 5.7 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the heterodimer formation was found assuming a pseudo first-order reaction kinetic. The equilibrium dissociation constant for homodimerization of ERalpha was 2.2 x 10(-8) to 5.4 x 10(-8) and 1.8 x 10(-8) to 2.6 x 10(-8)M for the heterodimer formation. The homo- and heterodimer formation was characterized by a slow association kinetics and kinetic rate constants were within the same range.     

Quantitative analysis of muscarinic acetylcholine receptor homo- and heterodimerization in live cells: regulation of receptor down-regulation by heterodimerization

Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo- and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M(1), M(2), and M(3) mAChR can form constitutive homo- and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M(1), M(2), or M(3) mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M(1)/M(2), M(2)/M(3), and M(1)/M(3) mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo- and heterodimers. When expressed in JEG-3 cells, the M(2) receptor exhibits much higher susceptibility than the M(3) receptor to agonist-induced down-regulation. Coexpression of M(3) mAChR with increasing amounts of the M(2) subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M(3), suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.     

Enhancement of the antagonistic potency of transforming growth factor-beta receptor extracellular domains by coiled coil-induced homo- and heterodimerization

Transforming growth factor-beta (TGF-beta) plays a causal role in several human pathologies including fibrotic diseases and metastasis. TGF-beta signaling is mediated through its interaction with three types of cell surface receptors, RI, RII, and RIII. The soluble ectodomains of RII and RIII bind to TGF-beta, making them attractive candidates to sequester TGF-beta and inhibit its activity. To optimize the activity of the ectodomains, we studied the effect of artificially dimerizing them upon their kinetics of binding to TGF-beta using an optical biosensor and studied their antagonistic potencies using an in vitro signaling assay. We fused the RII ectodomain and the membrane-proximal ligand-binding domain of the RIII ectodomain to de novo designed heterodimerizing coil strands and demonstrated that the coil strands within the fusion proteins were capable of promoting the dimerization of the coil-tagged ectodomains. Our results indicate that coiled coil-induced dimerization of the ectodomains stabilized their interaction with TGF-beta as compared with the monomeric ectodomains. Also, in contrast to the monomeric ectodomains, which did not block signaling, the coiled coil-induced dimers were characterized by antagonistic potencies in the low nanomolar range.     

Discrete simulation of regulatory homo- and heterodimerization in the apoptosis effector phase

MOTIVATION: Quantitative simulation of molecular reaction networks is among the most promising approaches towards an understanding of complex biochemical pathways. Numerous qualitative as well as quantitative data from diverse experimental settings, in particular from genomics and proteomics, have to be contextually linked to convert static data into dynamic functionality. RESULTS: This paper presents the Lattice Molecular Automaton, a Cellular Automaton-based simulation tool, capable of representing complex molecular dynamics at different levels of granularity. A data structure concept represents molecular units, whose dynamics, embedded on a 2D grid, is defined via detailed intermolecular interaction profiles. The data structures hold diverse information as molecular type, potential, as well as kinetic energy states, which allows a precise representation of intracellular reaction networks. The molecular dynamics is performed via local computation of individual molecular states on the lattice, which, in conjunction with discretized space and time, enables excellent scalability of this simulation concept. This paper finally gives Lattice Molecular Automaton simulation results on key elements of apoptosis, the cell death cascade, in particular focusing on the regulatory function of homo- and heterodimerization of members of the Bcl-2 protein family in the apoptosis effector phase. The regulatory proteins Bcl2, Bax, and Bak constitute a diffusion-driven molecular switch with inherent damping of apoptosis induction, thereby controlling the apoptosis reaction cascade under noisy, external apoptosis inducing conditions.     

Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer

Quantitative bioluminescence resonance energy transfer (BRET) analysis was applied to the study of beta(1)- and beta(2)-adrenergic receptor homo- and heterodimerization. To assess the relative affinity between each of the protomers, BRET saturation experiments were carried out in HEK-293T cells. beta(1)- and beta(2)-adrenergic receptors were found to have similar propensity to engage in homo- and heterotropic interactions suggesting that, at equivalent expression levels of the two receptor subtypes, an equal proportion of homo- and heterodimers would form. Analysis of the data also revealed that, at equimolar expression levels of energy donor and acceptor, more than 80% of the receptor molecules exist as dimers and that this high incidence of receptor dimerization is insensitive to receptor density for expression levels varying between 1.4 and 26.9 pmol of receptor/mg of membrane protein. Taken together, these results indicate that most of the receptors expressed in cells exist as constitutive dimers and that, at least in undifferentiated fibroblasts, the proportion of homo- and heterodimers between the closely related beta(1)- and beta(2)-adrenergic receptors is determined by their relative levels of expression.     

Calreticulin enhances B2 bradykinin receptor maturation and heterodimerization

In different native tissues and cells the receptor for the vasodepressor bradykinin, B₂, forms dimers with the receptor for the vasopressor angiotensin II, AT₁. Because AT₁/B₂ heterodimers may contribute to enhanced angiotensin II-stimulated signaling under pathophysiological conditions, we analyzed mechanisms of AT₁/B₂ heterodimerization. We found that efficient B₂ receptor maturation was a prerequisite for heterodimerization because only the fully mature B₂ receptor was capable to interact with AT₁. To identify chaperones involved in B₂ receptor maturation and heterodimerization we performed microarray gene expression profiling of human embryonic kidney (HEK293) cells. The expression of the chaperone calreticulin was up-regulated in cells with efficient B₂ receptor maturation. Vice versa, upon down regulation of calreticulin expression by RNA interference, B₂ receptor maturation and AT₁/B₂ receptor heterodimerization were significantly impaired. Concomitantly, the B₂ receptor-mediated enhancement of AT₁-stimulated signaling was reduced. Thus, calreticulin enhances B₂ receptor maturation and heterodimerization with AT₁.     

Oligomerization of the UDP-glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation

UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of beta-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.     

Modulation of homo- and heterodimerization of Harvey sarcoma virus RNA by GACG tetraloops and point mutations in palindromic sequences

Retroviruses harbour a diploid genome of two plus-strand RNAs linked non-covalently at the dimer linkage structure. Co-packaging of two parental RNAs is a prerequisite for recombination in retroviruses, but formation of heterodimers has not been demonstrated directly in vivo. Here, we explore elements in Harvey sarcoma virus (HaSV) RNA involved in homodimerization and heterodimerization with RNA of Moloney (Mo) and Akv murine leukemia viruses (MLV).By an in vitro assay, we found that HaSV dimerization specificity could be modulated by mutations in a decanucleotide palindrome (Pal) probably folded into a kissing-loop. Autocomplementary and non-autocomplementary sequences introduced into the putative loop directed the specificity towards formation of homodimers and heterodimers, respectively. Two stem-loop (SL) structures, both exposing a GACG tetraloop, enhanced the formation of stable HaSV dimers.A similar decanucleotide palindrome has been implicated in homodimerization of MLVs. Heterodimers between HaSV RNA and Mo- or Akv MLV were unstable, but could be stabilized by introduction of two point mutations in the putative HaSV kissing-loop, creating exact complementarity with Mo/Akv MLV palindromes. Moreover, such changes increased the HaSV RNA affinity for the two MLV RNAs. Similar to HaSV RNA homodimers, formation of heterodimers with Mo- or Akv MLV RNAs was induced by the presence of GACG loops.On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of gamma-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses.     

A possible mechanism for partitioning between homo- and heterodimerization of the yeast homeodomain proteins MATa1 and MATalpha2.

The yeast Saccharomyces cerevisiae has three cell types distinguished by the proteins encoded in their mating-type (MAT) loci: the a and alpha haploids, which express the DNA-binding proteins a1, and alpha1 and alpha2, respectively, and the a/alpha diploid which expresses both a1 and alpha2 proteins. In a/alpha cells, a1-alpha2 heterodimers repress haploid-specific genes and MATalpha1, whereas alpha2 homodimers repress a-specific genes, indicating dual regulatory functions for alpha2 in mating-type control. We previously demonstrated that the two leucine zipper-like coiled-coil motifs, called alpha2A and alpha2B, in the alpha2 N-terminal domain are important to a1-alpha2 heterodimerization. A unique feature of alpha2B is the occurrence of three atypical amino acid residues at a positions within the hydrophobic core. We have conducted mutational analyses of alpha2B peptides and the full-length protein. Our data suggest that these residues may play a critical role in partitioning of the alpha2 protein between heterodimerization with a1 and homodimerization with itself.     

Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A)

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst(2A) and sst(3) exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and sst(3). The sst(2A)-sst(3) heterodimer exhibited high affinity binding to somatostatin-14 and the sst(2)-selective ligand L-779,976 but not to the sst(3)-selective ligand L-796,778. Like the sst(2A) homodimer, the sst(2A)-sst(3) heterodimer stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst(2)-selective ligand L-779,976. However, unlike the sst(3) homodimer, the sst(2A)-sst(3) heterodimer did not promote GTPgammaS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst(3)-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst(2A)-sst(3) heterodimer desensitized at a slower rate than the sst(2A) and sst(3) homodimers. Both sst(2A) and sst(3) homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst(2A)-sst(3) heterodimer separated at the plasma membrane, and only sst(2A) but not sst(3) underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst(2A) and sst(3) results in a new receptor with a pharmacological and functional profile resembling that of the sst(2A) receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst(3) receptor function by heterodimerization with sst(2A) or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst(3)-specific binding and signaling in mammalian tissues.     

Mutations in retinoid X receptor that impair heterodimerization with specific nuclear hormone receptor

Retinoid X receptor (RXR) serves as a promiscuous heterodimerization partner for many nuclear receptors through the identity box, a 40-amino acid subregion within the ligand binding domain. In this study, we randomly mutated two specific residues within the human RXRalpha identity box region previously identified as important determinants in heterodimerization (i.e. Ala(416) and Arg(421)). Interestingly, most of these mutants still retained wild type interactions with thyroid hormone receptor (TR), retinoic acid receptor, peroxisome proliferator-activated receptor alpha, small heterodimer partner, and constitutive androstane receptor. However, RXR-A416D and R421L were specifically impaired for interactions with TR, whereas RXR-A416K lost both TR and retinoic acid receptor interactions. Accordingly, RXR-A416D did not support T3 transactivation in mammalian cells, whereas RXR-A416K was not supportive of transactivation by retinoids or T3. These results provide a basis upon which to further design mutant RXRs highly selective in heterodimerization, potentially useful tools to probe nuclear receptor function in vivo.     

A trafficking checkpoint controls GABA(B) receptor heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.     

Cannabinoid receptor type I modulates alcohol-induced liver fibrosis

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and 2 (CB1 and CB2) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, Δ9-tetrahydrocannabinol H?O?, endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H?O?, as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10 μmol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.